| Abstract | Oksidacijski stres se povezuje s patogenezom brojnih bolesti. U epidemiološkim studijama kao pokazatelj stupnja oksidacijskog stresa najčešće se koristi malondialdehid (MDA) koji nastaje kao produkt lipidne peroksidacije (LPO). Pretpostavlja se da je koncentracija MDA u urinu mjera oksidacijskog stresa cijelog organizma. Kako je urin lakše sakupiti od krvi svrha je ovog istraživanja bila razviti pouzdanu i primjenjivu metodu za mjerenje MDA u urinu. Za određivanje koncentracije MDA u urinu u ovom istraživanju koristila se metoda s 2-tiobarbituratnom kiselinom (2-TBA) u kojoj MDA reagira s dva ekvivalenta TBA i nastaje crveni adukt MDA-TBA2. Reakcija se odvija u kiselim uvjetima i na visokoj temperaturi. Smatra se da „stresni uvjeti“ reakcije (niski pH i visoka temperatura) dovode do dodatnog nastajanja MDA, odnosno do tzv. „lažno pozitivnih rezultata“. Ispitivani su optimalni uvjeti reakcijske smjese: različite temperature (80° i 90 °C) i različite dužine zagrijavanja (12, 20, 30, 40, 60 i 90 minuta) te različite koncentracije kiseline (0,1% i 1% o-H3PO4). Također ispitivane su i interferncije (urea i kreatinin). Ispitivanja su provedena na spektrofotometru Agilent 8453 UV-Vis (Agilent Technologies, CA, USA) na valnoj duljini od 532 nm. Pokazalo se da najoptimalnije pripremanje uzoraka uključuje 800 μl uzorka urina kojem se doda 500 μl 2-TBA (0.6%) i 1,5 ml o-fosforne kiseline (1%). Uzorci se potom promiješaju i zagrijavaju kroz 30 minuta na 90 °C u grijaćem bloku. Ispitivanje provedeno s ureom i kreatininom pokazalo je da one ne interferiraju u korištenim reakcijskim uvjetima. Za validaciju metode korišteni su standardi dobiveni razrjeđivanjem komercijalnog standarda MDA (1,1,3,3 tetraetoksipropan) koncentracija u rasponu od 0,76 do 20,23 μmol/L. Dobivena kalibracijska krivulja je bila pravac s jednadžbom y= 0,0562 x A + 0,0103 i koeficijentom korelacije R2= 0,9987. Ponovljivost izražena kao relativna standardna devijacija (RSD) bila je 1,38 %, a RSD ponovljivosti iz dana u dan 4,11 %. Limit detekcije bio je 0,107 μmol/L. Mjerenjem koncentracije MDA u 10 sakupljenih uzoraka urina dobrovoljaca dobivene su vrijednosti u rasponu od 1,69 do 5,92 μmol/L. To pokazuje da je razvijena metoda primjenjiva za mjerenje MDA u urinu te se može koristiti za određivanje koncentracije MDA u nepoznatim uzrocima odnosno za praćenje razine oksidacijskog stresa u urinu |
| Abstract (english) | Oxidative stress has been implicated in pathogenesis of many disease. In epidemiological studies as indicator of oxidative stress commonly used is malondialdehyde (MDA), formed as product of lipid peroxidation (LPO). It is assumed that concentration of MDA in urine is a measure of oxidative stress in the body as whole. Urine as a sample is much easier to collect then plasma. Therefore the aim of this study was to optimise method for determination of MDA concentration in urine. In this study for determination of MDA concentration is used thiobarbituric acid assay (TBA-assay) in which MDA reacts with two equivalents of 2-TBA and generate red adduct MDA-TBA2. This reaction is performed at acidic conditions and high temperature. It is considered that “stress conditions” in reaction (high temperature and low pH) can cause the formation of additional MDA, or so called false positive reactions. In this study the optimal temperature (80 °C and 90 °C), different heating intervals (12, 10, 20, 30, 40, 60, 90 minutes) and different acid concentrations (0,1% and 1% o-phosphoric acid) were investigated. Furthermore, interference (urea and creatinine) were investigated. This study was carried out on spectrophotometer Agilent 8453 UV-Vis (Agilent Technologies, CA, USA) at a wavelength of 532 nm. The results showed that the most optimal conditions in reaction mixture were 800 μL sample in addition of 500 μL 2-TBA and 1.5 mL of o-phosphoric acid. Samples where then mixed and heated for 30 minutes on 90 °C. Testing with urea and creatinine, as possible interferences, showed that they do not present interferences in reaction conditions. To validate the method series of MDA standards were prepared by diluting commercial standard (1,1,3,3 tetraethoxypropane) in the concentration range from 0.76 to 20.23 μmol/L. Calibration curve was linear with equation y= 0.0562 x A + 0.0103 and correlation factor was R2= 0.9987. Repeatability, expressed as relative standard deviation (RSD) was 1.38% and reproducibility RSD was 4.11 %. The detection limit was 0.107 μmol/L. The concentration of MDA in 10 urine samples collected from volunteers was in range from 1.69 to 5.92 μmol/L. This shows that developed method is suitable for detecting MDA in unknown urine samples in order to determinate oxidative stress. |
